(a) Field of Invention
The present invention relates to a glycopeptide isolated from human pituitary glands, viz., the N-terminal fragment of human pro-opiomelanocortin (POMC), and to a process for its preparation and purification.
(b) Prior Art
Although adrenocorticotropin (ACTH), beta-lipotropin (beta-endorphin (beta-END), and alpha- and beta-melanocyte-stimulating-hormone (alpha- and beta-MSH) are peptides with markedly different biological activities, yet they have been shown to be synthesized from a common precursor protein in mouse pituitary tumor cells, see e.g. Roberts et al., (1977) Proc. Natl. Acad. Sci. USA 74, 4826-4830, Mains et al., (1977) ibid. 74, 3014-3018, and Nakanishi et al. (1977) FEBS Letters 84, 105-109, and in the anterior and intermediate lobes of the mammalian pituitary, see e.g. Crine et al. (1978) Proc. Natl. Acad. Sci. USA 75, 4719-4723, Roberts et al. (1978) Biochemistry 17, 3609-3618, Hinnman et al. (1980) Biochemistry 19, 5395-5402, and Nakanishi et al. (1977) cited above. In spite of the fact that the two lobes seem to start with similar forms of the precursor, named pro-opiomelanocortin (POMC) see Chretien et al. (1979), Can. J. Biochem. 57, 1111-1121 who give an exhaustive review of the state of the art, they process these forms into different end products, see Mains et al. (1979) J. Biol. Chem, 254, 7885-7894, Seidah et al. (1978) Proc. Natl. Acad. Sci. USA 75, 3153-3157, Crine et al. (1979) Proc. Natl. Acad. Sci. USA 76, 5085-5089, and Roberts et al. (1978), Hinnman et al. (1980) and Chretien et al. (1979) cited above. Following excision of a 26 residues signal peptide, pulse-chase studies showed the initial maturation of pro-opiomelanocortin into beta-LPH and a large glycopeptide containing ACTH at its carboxy-terminus, see e.g. Gossard et al. (1980) Biochem. Biophys. Res. Commun. 92, 1042-1051, Seidah et al. (1980) Ann. N.Y. Acad. Sci. 343, 443-446, Herbert et al. (1980) Ann. N.Y. Acad. Sci. 343, 79-93. In the anterior lobe beta-LPH, ACTH and an N-terminal glycopeptide of hitherto unspecified C-terminal length are the end products of processing, see Gianoulakis et al. (1979) J. Biol. Chem. 254, 11903-11906, and Roberts et al. (1978) and Mains et al. (1979), both cited above. In the intermediate lobe, beta-LPH is processed further into gamma-LPH and beta-endorphin, see Crine et al. (1978) Roberts et al. (1978), Chretien et al. (1979), Mains et al. (1979), Seidah et al. (1978), Crine, et al. (1979), and Gianoulakis et al. (1979) all cited above, and ACTH is converted into alpha-MSH and possibly corticotropin-like-intermediate-lobe-peptide (CLIP), see Scott et al. (1973) Nature (London) New Biol. 244, 65-67, Scott et al. (1976) J. Endocrinol. 70, 197-205, and Mains et al. (1979), Crine, et al. (1979) and Gianoulakis et al. (1979), cited above. From pulse-chase experiments the fate of the N-terminal glycopeptide in this lobe seems to be similar to that of the anterior lobe, see Crine et al. (1980) Eur. J. Biochem. 110, 387-396, and Mains et al. (1979), Seidah et al. (1978) and Gianoulakis et al. (1979) cited above.
The advent of recombinant DNA technology has greatly facilitated studies of the nucleic acid sequence of genes coding for protein precursors. Using such techniques the mRNA sequence of bovine pars intermedia pre-pro-opiomelanocortin was first determined by Nakanishi et al. (1979) Nature 278, 423-427. This was followed by reports on the genomic DNA structures of human, bovine, and rat homologous sequences, see Chang et al. (1980) Proc. Natl. Acad. Sci. USA 77, 4890-4894, Nakanishi et al. (1980), Nature 287, 752-755, and Drouin (1980) Nature 288, 610-613, respectively. From these studies it became apparent that an MSH sequence exists in the N-terminal segment of pro-opiomelanocortin which bears sequence homology to both alpha- and beta-MSH. It was therefore called gamma-MSH by Nakanishi et al. (1979) cited above. Based on the results of pulse-chase experiments (see Crine et al. (1979), Gossard et al. (1980), Seidah et al. (1980) Herbert et al. (1980) Crine et al. (1980), and Gianoulakis et al. (1979), all cited above) the purification and the elucidation of the structures of the N-terminal fragment of human and of porcine pro-opiomelanocortin was attempted. While Hakanson et al. (1979), Nature 283, 789-792, reported on the primary sequence of the first 35 amino acid residues of the porcine homologue, Lariviere et al. (1980) FEBS Letters 122, 279-282, obtained a preliminary structure for the first 92 residues based upon tryptic peptide mapping and amino acid analysis. Concerning the human analog, Estevariz et al. (1980) Biochem. J., 191, 125-132, predicted the length of the peptide chain as about 82 amino acid residues based upon amino acid analysis, while Seidah et al. (1980) Biochem. Biophys, Res. Comm. 95, 1417-1424, established the sequence of the first 42 amino acids and showed by tryptic peptide mapping that the gamma-MSH region of the molecule was identical between species, see also Benjannet et al. (1980) Nature 285, 415-416. However, the exact length of the C-terminal sequence has hitherto still remained undetermined.
It has now been found that the N-terminal fragment of human pro-opiomelanocortin may indeed be prepared from fresh or frozen human pituitaries by the process of this invention and is thereby obtained in a substantially pure state which permits its characterization by chemical structure as an unambiguously established sequence of the 76 amino acids contained within its molecule. Furthermore, the nature of said N-terminal fragment as that of a glycopeptide, and the exact location of the glycosylation sites, as well as the presence and exact location of disulfide bridges are also established.